The process of lignin removal from chemical pulps to produce bright or completely white finished pulp is called ‘Bleaching’. It is necessary for aesthetic reasons and for improvement of paper properties, because the left over residual lignin after pulping imparts an undesirable brown color to paper. The present day bleaching of Kraft pulp uses large amounts of chlorine based chemicals. The use of these chemicals generates chlorinated organics, which being highly toxic cause various health hazards. Thus, an alternative and cost effective method i.e. the use of microbes and enzymes, has provided a very simple and economic way to reduce the use of chlorine and other bleaching chemicals.
Overview of the Various Organisms
Fungi
Lignin is the most abundant aromatic polymer in the biosphere. It is found in the cell wall of all vascular plants in association with cellulose and hemicellulose. Because inter-unit bonds in lignin are not hydrolysable, lignin is difficult to degrade either chemically or biologically. Lignin surrounds cellulose in the plant cell wall forming a matrix, which is itself resistant to degradation. Lignin biodegradation is responsible for much of the natural destruction of wood in use, and it may have an important role in plant pathogenesis. On the other hand, potential applications utilizing lignin-degrading organisms and their enzymes have become attractive, because they may provide environmentally friendly technologies for the pulp and paper industry. To date, only a few groups of organisms are capable of degrading complex lignin polymers, and they are best exemplified by the white rot fungi. Most of the research concerning biodegradation of lignin has been centered on some fungi only such as Phanerochaete chrysosporium, Streptomyces viridosporus, Pleurotus eryngii, Trametes trogii, Fusarium proliferatum (Regaldo et al., 1997) etc. (1)
Wood-rotting basidiomycetous fungi that cause white rot in wood are the most efficient lignin degraders in nature (Kirk and Farrell, 1987; Eriksson et al., 1990), and they are perhaps nature's major agents for recycling the carbon of lignified tissues. No other microorganisms as pure culture have been described to mineralize lignified tissues as efficiently (Kirk and Cullen, 1998). They are a group of taxonomically heterogeneous higher fungi, characterized by their unique ability to depolymerize and mineralize lignin using a set of extracellular lignnolytic enzymes. Lignin degradation by white-rot fungi has been intensively studied during the last thirty years in relation to biotechnical applications such as biopulping, biobleaching, treating of pulp mill effluents, and soil bioremediation (Akhtar et al., 1992, 1998; Lamar et al., 1992; Messner and Srebotnik, 1994).
In 1992, Frederick Archibald (2) demonstrated that the fungus, Trametes versicolor was capable of substantial decolorization and delignification of unbleached industrial kraft pulps over 2 to 5 days.
A year later, same group of workers demonstrated that a biobleaching culture supernatant contained all components necessary for hard wood Kraft pulp (HWKP) biobleaching and delignification, but the supernatant needed frequent contact with the fungus to maintain these activities. Thus, labile small fungal metabolites may be the vital Biobleaching system components renewed or replaced by the fungus. Nearly all the CO2 evolved by HWKP containing cultures came from the added glucose, indicating that HWKP is not an important source of energy during Biobleaching. The presence of HWKP in culture markedly increased the culture's production of a number of acidic metabolites, including oxalate, glyoxylate and glycolate.
Aspergilli, the versatile ascomycetes are also found to transform at a rapid rate a wide spectrum of lignin related aromatic compounds. They are shown to overproduce high levels of hemicellulolytic enzymes. (4)
Maria Teresa et al. have shown that Bjerkandera sp. Strain BOS55 is a white rot fungus that can bleach EDTA extracted eucalyptus oxygen delignified Kraft pulp (UKP) without any requirement for manganese. Furthermore, under manganese free conditions, addition of simple physiological organic acids (e.g. Glycolate, glyoxylate, oxalate and others) at 1-5 mM stimulated brightness gains and pulp delignification two to three fold compared to results not receiving acids. The stimulation was attributed to increase production of MnP and LiP as well as increased physiological concentrations of veratryl alcohol and oxalate. These factors contributed to greatly improved production of superoxide anion radicals, which may have been accounted for the more extensive biobleaching. (5)
Bacteria
The role of bacteria in lignin biodegradation is still a matter of conjecture. Some workers have demonstrated that either mixed (Sundman et al., 1968) or pure culture of bacteria (Sorensen, 1962) can grow on lignin as a carbon source. Pseudomonas spp. was claimed by Kawakami (1976) and Odier and Monties (1977) to degrade plant lignins. Odier and Montis also indicated several other bacterial strains that can use within seven days time more than 50% of the lignin supplied in a mineral medium containing glucose.
Several Nocardia and Pseudomonas spp. as well as some unidentified bacteria, isolated from lake water containing high loads of waste lignin, were tested for their capacity to release 14CO2 from specifically 14C-labelled dehydropolymer of coniferyl alcohol (DHP) or corn stack lignins. However only some of them could release significant amount s of 14CO2 from the labeled lignin. The tested Nocardia spp. was more active than the Pseudomonas spp. and the unidentified bacteria.(6)
Actinomycetes are filamentous bacteria which are found in soil and composts where lignocellulose is decomposed. Several reports provide evidence that several species belonging to the genus Streptomyces are able to degrade lignin. Other lignin degrading Actinomycetes include Thermomonospora mesophila, Actinomadura, Micromonospora with Streptomyces exhibiting the highest lignin degrading ability. In most of the studies, the lignin degrading enzyme was produced at higher levels in cultures containing lignocellulose which suggests that an induction mechanism was active.
Ajit Verma et al. (1994) while working on symbiotic relationship between termites and their intestinal microbes concluded that both termite soil and termite gut bacteria play an important role in polymer depolymerization. Gut bacteria have the capacity to degrade cellulosic and hemicellulosic materials more efficiently. Several bacterial isolates which hydrolyze cellulose and hemicellulose have been obtained in pure culture from the termite gut. Some of these are Arthrobacter sp., Bacillus cereus, Clostridium sp., Micrococcus sp., Streptomyces sp., Serratia marcescens. Only a few xylan decomposing bacteria have been obtained from the termite gut (Micrococcus luteuns, Pseudomonas aeruginosa). The question of lignin degradation by termites is intriguing, since much of the termite gut is anaerobic and natural anaerobic mechanisms of lignin degradation are unknown.(7)
Berrocal et al. (1997) have shown that cell free filtrates from streptomyces sp. Grown in solid state fermentation were capable of solubilising up to 20% of the [14C] lignin. The activity of two enzymes, extracellular peroxidase and phenol oxidase (laccase) was found to correlate with both solubilisation and mineralisation rates of lignin.(8)
The presence of bacteria in rotted wood often in association with fungi has been the subject of numerous reports. However, their exact role in degradation of wood components is still unclear. While the availability of nutrient nitrogen represses metabolism of synthetic 14C lignin to CO2 by Phanerochaete Chrysosporium, high levels of organic nitrogen were optimal for lignin degradation by the bacterium Streptomyces badius. (9)
Enzymes are the catalytic cornerstones of metabolism, and as such are the focus of intense worldwide research, not only in biological community, but also with process designers/engineers, chemical engineers, and researchers working in other scientific fields. Since ancient times, enzymes have played a central role in many manufacturing process, such as in the production of wine, cheese, bread etc. The latter half of the twentieth century saw an unprecedent expansion in our knowledge of the use of microorganisms, their metabolic products, and enzymes in a broad area of basic research and their potential industrial applications. Only in the past two decades, however have microbial enzymes been used commercially in the Pulp and Paper industry. (10)
The most common application of enzymes in paper industry is to enhance bleaching. At least 15 patents or patent disclosures dealing with enzymatic treatments to enhance bleaching of Kraft pulps were submitted between 1988 and 1993.
Kraft pulping, also known as sulphate, or chemical pulping, uses sulphur to get fiber out of trees. Kraft pulping uses less than 50% of the tree. The rest ends up as sludge which is burned, spread on land or land filled. A bonus of kraft pulping is that the chemicals can be recycled and re-used in the mill. Another is that kraft fiber is exceptionally strong (“kraft” means “strong” in German). Kraft pulp is usually dark and is often bleached with chlorine compounds.
The Kraft process accounts for 85% of the total pulp production in the United States, and it is the largest component of paper manufacture world-wide. Kraft pulping removes lignin, dissolves and degrades hemicellulose without damaging cellulose Unfortunately, degradation products generated during pulping become trapped in the matrix and impart a brown color to Kraft pulp. Cooking consumes pulping chemicals, and residual xylan (along with covalently-linked degradation products) precipitates on the surfaces of the cellulosic fibers. The chromophores are believed to be composed of residual lignin and carbohydrate degradation products. They are hard to extract because they are covalently bound to the carbohydrate moieties in the pulp matrix. Manufacturers use elemental chlorine (Cl2) and chlorine dioxide (ClO2) to bleach the chromophores, and then they extract the pulp to make white paper. The cost of Cl2 is about $12 to $15 per metric ton of pulp, but because this results in the production of chlorinated aromatic compounds, alternative-bleaching agents such as O2, ClO2 or H2O2 are employed. These can be several times more expensive than Cl2. Bleaching substantially increases the value of the product, so the additional expense can be justified.
Chlorine bleaching can create environmental problems. Byproducts from using these chemicals are chlorinated organic substances, some of which are toxic, mutagenic, persistent, and bioaccumulate, and cause numerous harmful disturbances in biological systems (Onysko, 1993). The available options are oxygen delignification, extended cooking, and substitution of chlorine dioxide for chlorine, hydrogen peroxide, and ozone. But most of these methods involve high capital investment for process change. Thus, an alternative and cost effective method, i.e. use of enzymes has provided a very simple and economic way to reduce the use of chlorine and other bleaching chemicals.
Till now, all the basics and applied research work has centered on fungi only. In case of biobleaching of raw pulp, the application of fungi is not feasible due to its structural hindrance caused by fungal hyphae. Bleaching of pulp using purified enzymes from fungi is not economical as the steps of enzyme purification make the process expensive and lengthy. Hence, there exists a need to develop an economically feasible and effective process for bio-bleaching of pulp.